DESCRIPTION: Most research on bacterial population genetics has focused nucleotide diversity and allelic variation generated by point mutations and recombination. In this proposal the investigator proposes a series of interesting studies to examine evolution of the genome organization and chromosomal structure in the divergence of bacterial strains. The overall objective is to address adaptive nature of major chromosomal organization and variation at the genomic level. The aims are to determine if bacterial genomes are under stabilizing selection such that bacteria with smaller chromosomes can accommodate greater amounts of extrachromosomal DNA, to study the organizational symmetry of the bacterial chromosome and determine if the occurrence of variable regions maintain the relative positions of the origin and terminus; and finally to examine the occurrence and genomic distribution of large blocks of foreign DNA which encode virulence determinants. To accomplish this the PI proposes to examine variation in the physical map of wild E. coli strains using restrictions digests and pulse gel electrophoresis. In preliminary studies, Dr. Ochman has demonstrated a wide range in genome sizes (4.7 to 5.3 Mb) among 15 natural isolates of E. coli (from the ECOR reference collection). A component of this variation in genome size is due to plasmid DNA and preliminary results suggest that there may be an inverse relationship between chromosome size and plasmid content. The first set of experiments will determine the plasmid content of natural E. coli isolates and separate the chromosome and plasmid components of genome size variation. The experiments involve isolation and digestion of plasmid DNA to identify plasmid fragments that contribute to genome size estimates. To identify parts of the chromosome that are subject to size variation, Dr. Ochman proposes to compare low resolution physical maps based on I-CeuI digestions. This restriction enzyme cleaves E. coli rRNA operons and typically yields 7 fragments from the entire chromosome. By comparing the fragment sizes among natural isolates, Ochman plans to identify regions of the chromosome that are susceptible to length mutations. The largest I-CeuI fragment (which encompasses about 1/2 of the E. coli chromosome) will be further analyzed by mapping BlnI restriction sites. Further delineation of the nature of the length variation will be accomplished through hybridization experiments using I-CeuI fragments (cut from PFG) and filters of the Kohara library. This analysis will identify which Kohara clones contain specific genes and gene regions missing from natural isolates. To test the hypothesis that natural selection maintains symmetry with regard to the origin and terminus of replication, Dr. Ochman proposes to complete studies of 36 E. coli, 2 Escherichia fergusoni strains, and 16 Salmonella strains for which detailed phylogenetic information is available. With the maps in hand, the investigator can statistically test for symmetry in chromosomal changes by locating the origin and terminus (by hybridization). Part of this phase of the work will focus on strains with known pathogenicity islands to see if symmetry has been restored by other chromosomal changes elsewhere in the genome. The study of chromosomal changes among E. coli and Salmonella strains in which phylogenetic information is available will allow this results of these detailed mapping experiments to be analysis in a phylogenetic context. Ochman further proposes to estimate the rates of chromosomal evolution by examining large scale changes in laboratory evolved E. coli that have be serially transferred for more than 10,000 generations (obtained from the laboratory of Richard Lenski).